近畿大学学術情報リポジトリ

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博士学位論文

Studies on development of the new method of control

using behavior regulators of Leptocorisa chinensis

(Dallas) (Hemiptera: Alydidae)

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博士学位論文

Studies on development of the new method of control

using behavior regulators of Leptocorisa chinensis

(Dallas) (Hemiptera: Alydidae)

(クモヘリカメムシの行動制御物質を使った新たな防除法の研究)

平成 29 年 5 月 17 日

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Nymphs

Pre-ovispoition

(℃) d (mean±SD)

n

a

_{d (mean±SD)}

_n

a

_{d (mean±SD) n}

a

18 _{14.9 ± 2.6}

89 47.1 ± 1.9

81 29.5 ± 3.6

14

22 _{10.4 ± 1.2}

34 31.5 ± 1.7

29 21.1 ± 2.3

11

25 _{8.7 ± 0.7}

250 25.2 ± 2.5

142 17.3 ± 5.3

14

30 6.7 ± 0.7

197 18.7 ± 2.1

172 12.4 ± 3.2

20

a _{Number of individuals tested.}

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by the linear regression equation (Y=a×bT), where Y is the reciprocal of the number of

days (= development rate) and T is temperature (°C) (Patel and Schuster, 1983). There

was a significant linear relationship between temperature and development rates of eggs,

nymphs and pre-oviposition. The developmental zeros in eggs and nymphs were at 8.1

and 10.1°C, respectively, and the effective heat units were 147 day-degrees and 370

day-degrees, respectively. The developmental zero and effective heat unit for the

pre-oviposition period of nondiapause females were 9.6°C and 256 day-degrees,

respectively.

Table 1-2. Regression equations of developmental rate (Y) to rearing temperature (T) in Leptocorisa chinensis, developmental zero and total effective heat units calculated from the regression equation

Stage Regression equation r2 Developmental zero Total effective heat units

(°C) (day-degrees) Egg Y=0.0068T-0.0552 0.99** 8.1 147 Nymph Y=0.0027T-0.0274 0.99** 10.1 370 Pre-oviposition period Y=0.0039T-0.0374 0.99 ** _9.6 ₂₅₆ **ｐ<0.01

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The cumulative percentage of ovipositing females collected on 9 November 2001 and

12 February 2004 gradually increased (Fig.1-1). All females had laid their eggs by 41 d

after incubation. There was no significant difference in the days when egg laying started

after incubation between the populations collected on 9 November 2001 (28.6 d±5.7

SD) and 12 February 2004 (33.3 d ±4.9 SD) (p>0.05, t-test). From these results, an

effective heat unit of 469.7 day-degrees above 9.6°C, which is the threshold for females

of non-reproductive diapause, was assumed to be equal to that of reproductive diapause,

and was estimated to be required for the pre-oviposition period of overwintering

females. 0 20 40 60 80 100 1 6 11 16 21 26 31 36 41 46 C um ul at iv e % of ov iposi ti on

Day after incubation (25ºC，16L-8D)

Fig. 1-1. Pre-oviposition period of female, Leptocorisa chinensis transferred from the field to laboratory conditions. Solid and dashed lines indicate females collected on 9 November 2001 and 12 February 2004, respectively.

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Discussion

In L. chinensis, Hasegawa et al. (1976) reported that this bug overwinters as adults.

In the present study, we recognized that all the adult females collected in winter

gradually began to lay eggs after incubation (Fig. 1-1). An effective heat unit of 469.7

d-degrees was required for the pre-oviposition period of overwintering females,

although the effective heat unit for the pre-oviposition period of undiapaused females

was 256 day-degrees. We have never observed the level of egg maturation in

overwintering females, but Hasegawa et al. (1976) observed that the females have no

mature eggs in the winter. The same genus, Leptocorisa oratorius Fabricius is known to

have reproductive diapause (Ito et al. 1993). It is considered that adult L. chinensis

would undergo reproductive diapause in the winter. There was no significant difference

in the timing of egg laying after incubation between the populations collected on 9

November 2001 and 12 February 2004. It is thought that the level of ovary maturity

between both of the populations is the same. Since there was not much difference, the

effective heat unit for the pre-oviposition period of L. chinensis females in the

meteorological data for November to February from 2001 to 2004 was recorded with

the AMeDAS system in Kasai City, Hyogo Prefecture. A more intensive experiment

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Here, the number of annual generations of L. chinensis was estimated based on

these data and the meteorological data from 2001 to 2003 recorded by the AMeDAS

system in Kasai City, Hyogo Prefecture. The starting point for the calculation was 1

January. The results indicated that adults of the first generation and second generation

emerged in mid-July and late August, respectively (Fig. 1-2). The possibility also

showed that adults of the third generation would appear depending on temperature. In

0

250

500

750 1000

Ef fect iv e cu m u lat iv e te m p e ratu re (da y -d eg re e)

J F M A M J J A S O N

D

Preoviposition period

Nymph

Egg

OW

G1

_G2

G3

0

250

500

750 1000

Ef fect iv e cu m u lat iv e te m p e ratu re (da y -d eg re e)

J F M A M J J A S O N

D

Preoviposition period

Nymph

Egg

OW

G1

_G2

G3

Fig. 1-2. Schematic seasonal advancement of Leptocorisa chinensis generations from 2001 to 2003 in Hyogo Prefecture, estimated from the developmental zero and effective heat units of eggs, nymphs, and the pre-oviposition period. OW: Overwintering generation, G1: First generation, G2: Second generation, G3: Third generation. 2001: solid lines, 2002: broken lines, 2003: single dotted lines.

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the earlier study, two peaks were observed in July and September in Shiga Prefecture

(Hasegawa et al., 1976), and also in Hyogo Prefecture (Yamashita, unpublished) in the

Kinki region. The present results are consistent with these reports, in that the number of

annual generations of L. chinensis was two.

Earlier studies investigated the effects of constant temperature on the rate of

development of L. chinensis reared on unhulled rice in milky stages every 7 d (Ishizaki

et al., 2002). Developmental thresholds of 13.5, 12.0 and 18.0°C, and thermal constants

of 92.4, 316.2 and 247.7 d-degrees were estimated for the eggs, nymphs and

pre-oviposition period, respectively. Developmental rates obtained from those results

did not provide a good estimate of the developmental times of populations in the field.

A direct extrapolation of the results to the field did not fit the field fluctuation of L.

chinensis. The development of L. chinensis reported by Ishizaki et al. (2002) was longer

than that in the present results. The estimated lower developmental threshold

temperatures recorded in the present study differed slightly from those estimated using

the results of Ishizaki et al. (2002). These differences may be due to differences in the

quality and quantity of food materials the tested insects consumed or/and differences in

developmental thresholds between the geographic populations of L. chinensis from

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These results would be useful to estimate the occurrence of each stage of L.

chinensis. However, to apply the developmental simulation to L. chinensis, further

studies are needed for the measurement of another developmental parameter as well as

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Chapter 2

Temperature and photoperiodic effects on induction and termination of diapause

in female Leptocorisa chinensis

Introduction

In Chapter 1, the developmental threshold temperature (9.6°C) and the heat unit

requirement (469.7 day-degrees) during the preoviposition period of female L. chinensis

were estimated. It was also estimated that there are two or three annual generations.

However, the field population phenology of L. chinensis was predicted from these

results without considering the consequences of diapause. Both the induction and the

termination of diapause in insects can influence the seasonal timing of growth and

reproduction in the generation immediately after diapause and in subsequent generations

(Tauber et al. 1986). Therefore, it is important to understand diapause induction and

termination in L. chinensis to determine the field phenology. In a recent study,

Tachibana and Watanabe (2007) reported that adult L. chinensis collected from Tsukuba

(36.02°N, 140.10°E), Ibaragi Prefecture, Japan, undergo a winter reproductive diapause,

and the critical day length for diapause induction was 13.75 h/d. However, they did not

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one of the sensitive stages of insects that undergo reproductive diapause (de Wilde 1954,

Tauber and Tauber 1976, Numata 2004, Danks 2007). In Japan, nymphal and adult L.

chinensis occur in the fields in autumn when the day length is shorter than 13.75 h

(Takeuchi et al. 2005, Yamashita et al. 2005, Tachibana and Watanabe 2007).

This chapter investigated the influence of photoperiod and temperature on the

induction and termination of reproductive diapause in female nymphal and adult L.

chinensis. These results contribute to understanding the life cycle of L. chinensis in the

field and to the establishment of integrated management strategies to counteract this

major pest to rice.

Temperature and photoperiodic effects during nymphal and adult stages on

female L. chinensis survival and oviposition

A culture of L. chinensis was established from adults collected in a paddy field

and neighborhood sites in Kasai (34.55°N, 134.59°E), Hyogo Prefecture, Japan, during

October 2005. Before experimentation, the insect culture was maintained for two

generations at 25 ± 0.5°C under a photoperiod of 16:8 (L:D) h in a plastic cage (9 cm

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distilled water for drinking (Yamashita, 2010). Randomly selected, newly emerged

nymphs (<1 d old) from the third filial generation were maintained under either a

constant long-day (16:8 [L:D] h) or short-day (12:12 [L:D] h) photoperiod at 25±0.5°C.

Adults that emerged from each photoperiod group (<1 d old) were then randomly

allocated to one of the two photoperiods at 25±0.5°C, 20±0.5°C, or 15±0.50.5°C, for a

total of 12 experimental adult groups. The initial number of females per group was 11－

24. All adults were reared as male-female pairs in plastic cups (6 cm dia.×4 cm height)

sustained on frozen, then defrosted, milk-ripe stage rice, along with distilled water.

Mortality and the number of females that oviposited were recorded daily for 100 d.

Fisher exact probability test was used to determine the significance of the differences in

the survival rate among the different conditions.

Photoperiodic effects on survival and postdiapause reproductive recovery of

female L. chinensis

L. chinensis- cultures were established from adults collected from a wild grass

habitat near a paddy field in Kasai, Hyogo Prefecture, Japan, on 8 December 2005.

Until experiments began, they were maintained in an outdoor cage (9 cm dia.×5 cm

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overwintering adults were transferred to the laboratory for experiments on 1 February

and 29 March 2006, where they were randomly allocated into male-female pairs and

maintained under either a constant long-day (16:8 [L:D] h) or short-day (12:12 [L:D] h)

photoperiod at 25±0.5°C and sustained on defrosted frozen rice in the milk-ripe stage

and distilled water for the duration of the experiment. The initial number of females was

10-12 per cohort. For comparison, field-collected adults from Kasai were transferred to

the laboratory on 10 June 2005 and maintained under either a constant long-day (16:8

[L:D] h) or short-day (12:12 [L:D] h) photoperiod at 25±0.5°C. Mortality and the

number of females that oviposited were recorded daily for the duration of the

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Results

Temperature and photoperiodic effects during nymphal and adult stages on female

L. chinensis survival and oviposition

The effects of temperature and photoperiod on survival and oviposition of adults

reared as nymphs under a long-day photoperiod (16:8[L:D] h) are shown in Fig. 2-1. L.

chinensis females started ovipositing on day 11 after adult emergence at 25°C under a

long-day photoperiod, and >60% of the females oviposited during the experimental

period (Fig. 2-1a). At 20°C under a long-day photoperiod, adults started ovipositing on

day 21 after adult emergence, and the number of ovipositing females increased

gradually (Fig. 2-1c). Females under a long-day photoperiod at 15°C laid no eggs,

although they survived for the duration the experimental period (100 d; Fig. 2-1e).

Females under a short-day (12:12 [L:D] h) photoperiod at 25°C started ovipositing on

day 10 after adult emergence. They ceased ovipositing within a few days but survived

for the duration of the experimental period (Fig. 2-1b). Females under a short-day

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Fig. 2-1. Effects of temperature and photoperiod on survival and oviposition of adult female L. chinensis reared as nymphs under a long-day photoperiod of 16:8 (L:D) h under 25°C (a and b) , 20°C (c and d), and15°C (e and f). Solid and broken lines represent the percentage of adult female surviving and ovipositing, respectively, The initial number of newly emerged females (<1 d old) was 11-24 (n).

Days after transfer

e 15°C n=12 f 15°C n=12 d 20°C n=11 c 20°C n=18 b 25°C n=24 16L- 8D 12L-12D a 25°C n=18

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The effects of temperature and photoperiod on survival and oviposition of adults

that were reared as nymphs under a short-day photoperiod (12:12 [L:D] h) are shown in

Fig. 2-2. Females under a long-day photoperiod (16:8 [L:D] h) at 25°C started

ovipositing on day 40 after the start of the experiment and the number of ovipositing

females increased gradually (Fig. 2-2a). Under a long-day photoperiod at 20°C, a small

number of females oviposited (Fig. 2-2c). However, no females oviposited under any

other conditions (Fig. 2-2e and f).

There were no significant differences in the survival rate at day 100 between the

females in each photoperiodic condition in the same temperature in Figs. 2-1 and 2 (P>

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Fig. 2-2. Effects of temperature and photoperiod on survival and oviposition of adult female L. chinensis reared as nymphs under a short-day photoperiod of 12:12 (L:D) h under 25°C (a and b), 20°C (c and d), and 15°C (e and f). Solid and broken lines represent the percentage of adult female surviving and ovipositing, respectively. The initial number of newly emerged females (< 1 d old) was 11-24 (n).

Days after transfer

e 15°C n=12 f 15°C n=11 d 20°C n=11 c 20°C n=12 b 25°C n=12 16L- 8D 12L-12D a 25°C n=12

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Photoperiodic effects on survival and postdiapause reproductive recovery of

female L. chinensis

Adult females transferred from the overwintering cage in the field to the long-day

photoperiod at 25°C in the laboratory on both 1 February and 29 March commenced

ovipositing day ≈40 after transfer (Fig. 2-3a and c). However, no adult females that

were transferred on either date to the short-day photoperiod at 25°C oviposited for the

duration of the experiment (150 d; Fig. 2-3b and d). Field-collected adult females that

were transferred to a long-day photoperiod at 25°C in the laboratory on 10 June

commenced oviposition almost immediately and continued to lay eggs for the duration

of the experiment (Fig. 2-3e). Similarly, collected adult females transferred to a

short-day photoperiod at 25°C in the laboratory on the same day commenced

ovipositing immediately. However, after 14 d the number of ovipositing females

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Fig. 2-3. Survival and oviposition of adult female L. chinensis transferred from field conditions to the laboratory and maintained at 25°C and either a 16:8 (L:D) h (left) or 12:12 (L:D) h (right) photoperiod. Solid and broken lines represent the percentage of adult female surviving and ovipositing, respectively. The initial number females was 10-12 (n). Survivorship and oviposition data reported in a-d are for adults collected on 8 December 2005 and in e and f for adults collected on 10 June 2005. Dates when adults were transferred to the laboratory conditions are shown.

Days after transfer

b 1 Feb. n=11 a 1 Feb. n=10 e 10 Jun. n=12 f 10 Jun. n=11 d 29 Mar. n=11 c 29 Mar. n=10

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Discussion

Nymphal and adult stages are the most sensitive to photoperiod and temperature

in many insects that undergo reproductive diapause (de Wilde 1954, Tauber and Tauber

1976, Numata 2004, Danks 2007). In L. chinensis, oviposition curves differed between

similarly maintained adults that had been reared as nymphs under differing photoperiod

conditions (Figs. 2-1 and 2). Likewise, oviposition was influenced by differing

photoperiod conditions during the adult stage of nymphs that were reared under the

same conditions (Figs. 2-1 and 2). More specifically, adult females that had been reared

as nymphs under the long-day photoperiod oviposited for only a short time under

short-day conditions at 25°C (Fig. 2-1b). Females did not oviposit under a short-day

photoperiod at 20°C, even when they were reared as nymphs under the long-day

photoperiod (Fig. 2-1d). These findings suggest that short-day photoperiod conditions

during the adult stage induce diapause in L. chinensis females. This is consistent with

the findings of Tachibana and Watanabe (2007).

There were also differences in oviposition patterns between the adult stages of L.

chinensis nymphs reared under the long-day and short-day photoperiods (Fig. 2-1a and

c , Fig. 2-2a and c). Adults began ovipositing after ≈40 d under a long-day photoperiod,

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Females required constant long-day photoperiod conditions to initiate oviposition,

indicating that in L. chinensis, not only adult but also nymphal stages are sensitive in the

process of reproductive diapause induction. Thus, both adults and nymphs can undergo

a reproductive diapause. This is further supported by the fact that in Japan, both nymphs

and adults can be found in the field when the daylength is shorter than the critical

photoperiod of 13.75 h/d. (Takeuchi et al. 2005, Yamashita et al. 2005, Tachibana and

Watanabe 2007). Thus, it was concluded that L. chinensis females exhibit a facultative

adult diapause and that photoperiod controls its’ induction and termination, confirming

the results of Tachibana and Watanabe (2007).

There were also differences in oviposition patterns between the adult stages of L.

chinensis nymphs reared under the long-day and short-day photoperiods (Figs.2-1a and

c and 2a and c). Adults began ovipositing after ≈40 d under a long-day photoperiod,

when they had been reared as nymphs under a short-day photoperiod (Fig. 2-2a and c).

Females required constant long-day photoperiod conditions to initiate oviposition,

indicating that in L. chinensis, not only adult but also nymphal stages are sensitive in the

process of reproductive diapause induction. Thus, both adults and nymphs can undergo

a reproductive diapause. This is further supported by the fact that in Japan, both

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critical photoperiod of 13.75 h/d. (Takeuchi et al. 2005, Yamashita et al. 2005,

Tachibana and Watanabe 2007). Thus, it was concluded that L. chinensis females

exhibit a facultative adult diapause and that photoperiod controls its’ induction and

termination, confirming the results of Tachibana and Watanabe (2007).

The results of the transfer experiments also indicate L. chinensis females exhibit a

facultative adult diapause. For example, when transferred to 25°C in the laboratory,

field overwintering adult females did not commence oviposition when maintained under

short-day conditions but commenced ovipositing after ≈40 d under long-day

photoperiod conditions (Fig. 2-3). Moreover, field-collected, post overwintering (June)

female adults maintained under short-day conditions did not oviposit as readily or as

consistently as those maintained under the long-day photoperiod at 25°C (Fig. 2-3).

The termination of diapause in L. chinensis might also be infuenced by

temperature. When females were reared as nymphs under a short-day photoperiod, and

maintained as adults under a long-day photoperiod, oviposition began after ≈40 d, but

the percentage that oviposited over the experimental period of 100 d was lower at 20°C

than at 25°C (Fig. 2-2a and c). Females did not lay any eggs at 15°C regardless of the

photoperiod conditions. Tachibana and Watanabe (2007) assumed that reproductive

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longer than the critical photoperiod and when temperatures exceeded a given high,

although females never began oviposition during the 30-d experiments. In our study,

field overwintering adult females transferred to 25°C in the laboratory did not

commence oviposition when maintained under short-day conditions but did so under

long-day conditions after ≈40 d (Fig. 2-3a and c). These results suggest that, to

commence oviposition after winter, female L. chinensis require a long-day photoperiod

and ≈40 d at temperatures >20°C.

Oviposition patterns for the 1 February cohort were basically the same as those for

the 29 March cohort when they were transferred to both short-and long-day photoperiod

conditions (Fig. 2-3a and d). These results indicate that changes in photoperiod or

temperature did not terminate diapause in L. chinensis females by 29 March. However,

by 10 June, adult female L. chinensis were able to oviposit readily when transferred to

long-day photoperiod conditions and had thus recovered their reproductive ability after

diapause. The females also oviposited for a short period when they were transferred to

short-day photoperiod conditions, but the number of ovipositing females gradually

decreased (Fig. 2-3f). This pattern was similar to that of adult females reared as nymphs

under a long-day photoperiod and then held under short-day photoperiod at 25°C (Fig.

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Additional studies are needed to evaluate the developmental parameters for field

populations of L. chinensis in various climatic regions. Furthermore, in the current study,

the diapause of L. chinensis was evaluated using oviposition and survival data without

examining the condition of the ovaries and fat body. To get more information about

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Chapter 3

A pecky rice-causing stink bug Leptocorisa chinensis escapes from volatiles emitted

by excited conspecifics

Introduction

Disturbed stink bugs emit pungent volatile compounds that could have several

ecological functions including behavior regulation and defense against predators (Blum

1985; Aldrich 1995). The behavior regulator function might be used for the behavioral

control of stink bugs in agroecosystems. The volatiles from smashed bugs likely repel

other stink bugs in the area. We hypothesized that the volatiles emitted from disturbed L.

chinensis could be used to repel conspecifics in rice fields and reduce the number of

pecky rice grains.

Leal et al. (1996) identified the volatiles emitted from L. chinensis anaesthetized

with CO2to minimize the release of defensive secretions, and found that a 5:1 mixture

of (E)-2-octenyl acetate and octanol, major components of the emissions of undisturbed

L. chinensis was an attractant pheromone. However, the chemistry and ecological

functions of the volatiles from disturbed L. chinensis individuals have not yet been

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The objective of this study was to clarify whether the volatiles from disturbed L.

chinensis elicit excitement and escape behavior in conspecifics as a first step to test the

above hypothesis. We first investigated whether L. chinensis escaped from disturbed

conspecifics in an observation arena under laboratory conditions. We then analyzed the

volatiles emitted by both disturbed and undisturbed L. chinensis and observed the

responses of undisturbed L. chinensis to the components in the volatiles from disturbed

conspecifics.

Materials and methods

Insects

A colony of L. chinensis was established from adults collected from paddy fields

in Kasai, Hyogo Prefecture, Japan, in October 2005, and maintained for two generations

prior to the experiments. Males and females were kept in a plastic cage (9 cm dia.×5 cm

height) in a climate controlled room at 25 ± 0.5°C under a photoperiod of 16L:8D, with

distilled water in a small Petri dish and rice panicles (ca. 50 mm) in the milk-ripe stage

as food. After mating occurred in the cage, I used them for the experiments. For

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Fig. 3-1. Bioassay set-up to evaluate the responses of Leptocorisa chinensis to alarm odours . An undiaturbed adult was introduced into an observation arena made from two plastic cups (85mm diameter 40mm height), and then either (A) an L.chinensis or (B) a piece of filter paper imoregnated with a synthetic chemical was introduced through the hole (18mm diameter).

Observation arena An excited L. chinensis

Rice grains for food A piece of filter paper with sample A plastic cup

Wet cotton towel for water supply Hole (5mm diam.) A calm L.chinensis (ca.1.5cm long) Hole (18mm diam.) 8.5cm 4 cm water ( A ) ( B )

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Bioassays

The bioassay set-up was constructed to evaluate the excitement and escape

behaviour of undisturbed L. chinensis to cues from excited conspecifics (Fig. 3-1).

Water (60 cc) was provided in a lidded plastic cup (85 mm diameter, 40 mm height).

The lid had a 5-mm-diameter opening, into which a piece of wet cotton towel was

inserted. I placed 10 rice seeds at the milk-ripe stage on top of part of the lid as food. I

covered the lid with an inverted plastic cup of the same size with one escape hole (18

mm diameter) at the side. I called the inside of the inverted plastic cup the observation

arena.

To introduce an undisturbed individual into the arena, we carefully transferred one

L. chinensis from a rearing cage to a test tube (18 mm diameter, 120 mm length), and

the opening of the test tube was connected to the opening in the inverted plastic cup to

allow the bug (initial occupant) to enter the arena. When the bug stopped moving, it was

considered undisturbed. For the control experiment, we introduced an undisturbed bug

into the arena in the same way. For experimental treatments, we introduced an

individual of the same gender using forceps with gentle nipping to disturb it and cause it

to emit the pungent odour. The two individuals were distinguishable because the second,

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initial occupant was observed for 3 min. When the occupant raised its antennae to scan

the air and started walking actively, it was judged to be excited. When it left the arena

through the hole, we judged that it had escaped. We measured the duration of time until

it showed excitement and escape behaviour. The introduced disturbed individual was

actively walking in a circle on the ceiling of the arena. During the observation, the two

individuals did not interact in the arena.

To further test the ecological functions of the pungent volatiles from L. chinensis,

I conducted the same bioassays using the dominant chemical components. A pure

compound (1 or 10 μg) was applied to a piece of filter paper (10×10 mm2_{) and}

introduced into the observation arena containing an undisturbed L. chinensis through the

opening (Fig. 1-1). The amounts of pure compound [(E)-2- octenal] were determined

based on chemical analyses: the amounts released by one disturbed female per minute

were ca. 4.5–14μg . We measured the duration of time until the bug showed excitement

and escape behaviour. The bioassays were conducted in a climate-controlled room (25 ±

3°C, 50–60% relative humidity; during 10:00–16:00). I repeated each experiment, 5–10

times per day on 2–4 experimental days.

Then the responses of males and females of L. chinensis were tested to

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to excite the bugs. In this study, we needed (E)-2-octenal to volatilise slowly to measure

the air concentrations, so we dissolved (E)-2-octenal in methanol (10% v/v) and then

diluted with distilled water to make 0.1, 0.01, and 0.001% solutions. For each bioassay,

we used 1 mL solution impregnated into a piece of moist cotton wool (2 cm×2 cm×0.5

cm) on the same size of aluminium foil. We conducted experiments in a mesh cage (30

×30 × 50 cm3: 2 mm mesh) with an electric fan (flow rate 50 cm/s; SY124010L, 40 mm

× 30 mm × 10 mm thickness; Size Corporation, Tokyo, Japan) in the centre of the cage.

We carefully introduced 4–5 undisturbed L. chinensis (either males or females) into the

mesh cage as described above. When the bugs stopped moving, they were considered

undisturbed. We then placed a piece of impregnated cotton wool on the fan. We

observed the flight behavior for 5 min. In our preliminary behavioural observations of

both sexes in the mesh cage, we confirmed that flying individuals did not elicit any

behavioural responses in undisturbed individuals. For both males and females, we

conducted the experiments 4 times (0.1% solution and 0.01% solution) and 3 times

(0.001% solution) on 1–3 experimental days in a climate-controlled room (25 ± 3°C,

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Volatile collection and chemical analysis

Either a male or a female L. chinensis was used for volatile sampling. For

collection from a disturbed individual, it was nipped with forceps. Immediately

afterwards, it was put in a 200-mL glass bottle with an air inlet and outlet (55 mm

diameter, 110 mm height). For the collection of volatiles from an undisturbed individual,

it was allowed to walk into the volatile collection vial from the rearing cage. An

undisturbed individual was motionless, walking, or flying in the bottle. Volatile

collections were done on four individuals per gender and treatment during daytime

(25±3°C, 50–60 % relative humidity; during 10:00–15:00).

The headspace volatiles in the glass bottles were collected for 1 min (disturbed

adult) or 10 min (undisturbed) at flow rate of 100 mL/min using Tenax adsorbent in a

glass tube (Tenax TA 20/35 100 mg; 3-mm inner diameter (ID), 160 mm long; GL

Science, Tokyo, Japan). The volatile collection time was determined based on

preliminary chemical analyses. The trapped compounds from disturbed adults were

eluted with 2 mL n-hexane (Wako Pure Chemical Industries Ltd.) containing n-eicosane

(0.5 μg; Wako Pure Chemical Industries Ltd.) as internal standard for the recovery rate.

The eluate was concentrated by nitrogen gas flow to 10 μl. One microliter of

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38

chromatograph–mass spectrometer (GC–MS; GC: Agilent 6890 with HP-5MS capillary

column: 30 m long, 0.25 mm i.d. and 0.25 μm film thickness; MS: Agilent 5973 mass

selective detector, 70 eV with He as carrier gas; Agilent, Santa Clara, CA, USA). The

GC oven temperature was programmed to rise from 40°C (5 min hold) to 280°C at

15°C/min. The compounds were identified by comparing their mass spectra and

retention times with those of authentic compounds and quantified using a calibration

curve of the respective compound.

Air in the flight cage with volatilised (E)-2-octenal was collected for 3 min at flow

rate of 100 mL/min using Tenax adsorbent in a glass tube [Tenax TA 20/35 100 mg;

3-mm inner diameter (ID), 160 mm long]. The collections were repeated four times for

each of the four concentrations. The volatile collection time was determined based on

preliminary chemical analyses. The collected volatile compounds were analysed by

GC–MS as described above, except for the injection method. The GC–MS was

equipped with a thermal desorption cold trap injector (TCT; CP4010, Chrompack, The

Netherlands). Headspace volatiles collected on Tenax-TA were released in the TCT

thermal desorption unit at 220°C for 8 min in He flow. The desorbed compounds were

collected in the TCT cold trap unit (SIL5CB-coated fused silica capillary) at -130°C.

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39 capillary column of the GC.

Chemicals

Hexyl acetate, octyl acetate, octanal, (E)-2-octenal and octanol were purchased

from Wako Pure Chemical Industries Ltd., Osaka, Japan. (E)-2-Octenyl acetate and

(Z)-2-octenyl acetate were synthesised by Sumika Technoservice Corporation (Hyogo,

Japan).

Statistical analyses

The amounts of each volatile compound emitted from males and females were

compared by t test. The time durations needed for excitation and escape were tested by

the Kaplan–Meier time-to-event model using a log-rank test statistic. All statistical tests,

except multiple-comparison tests, had a significance of 0.05. For multiple comparisons

of duration, we adjusted the significance according to Holm’s sequentially rejective

Bonferroni test to reduce type I errors. The multiple comparisons involved three

log-rank tests, therefore the lowest of the three P values was compared with a = 0.0167

(0.05/3), the second lowest with α = 0.025 (0.05/2), and the third lowest with α = 0.05

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40

(E)-2-octenal were analysed using two-way analysis of variance (ANOVA) with factors

concentration and sex, and their interaction. The data of the flight proportion were

arcsine square root transformed before two-way ANOVA, and were weighted in the

analysis by the number of individuals released into the mesh cage. All statistical

analyses were conducted using the JMP software package (version 9.0.2; SAS Institute,

Cary, NC, USA).

Results

Response of L. chinensis to volatiles emitted from conspecifics

Following the introduction of a disturbed same-sex conspecific, all undisturbed

females (n = 19) and males (n = 16) exhibited excitement within 40 s (Fig. 3-2a) and

escaped the arena within 3 min (Fig. 3-2b), with no differences between the sexes (P =

0.14 and P = 0.72 log-rank test, respectively). When an excited individual touched the

hole (18 mm diameter) with antennae, the individual immediately walked out of the

arena through the hole and flew to the fluorescent lights on the ceiling. In contrast,

addition of an undisturbed same-sex conspecific elicited no excitement or escape

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41

Chemical analyses of volatiles emitted from L. chinensis

females with a disturbed female

Undisturbed males with a disturbed male

Undisturbed females with an undisturbed female males with an undisturbed male

Fig. 3-2. Proportions of excitement and escape of undisturbed Leptocorisa chinensis females (n = 19) and males (n = 16) when exposed to undisturbed or disturbed conspecifics of the same gender. Lines with the same lower-case letter are not significantly different (Holm’s sequentially rejective Bonferromi test after Kaplan-Meier time-to-event model using a log-rank test statistic, P<0.05). Females (n = 19) and males (n = 16) exposed to an undisturbed individual showed no excitement/escape behavior.

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42

Chemical analyses of volatiles emitted from L. chinensis

Volatiles from undisturbed males and females were below detectable levels (data

not shown). When disturbed, both male and female L. chinensis adults (n = 4) emitted

hexyl acetate, octyl acetate, (Z)-3-octenyl acetate, (E)-2-octenyl acetate, octanal,

(E)-2-octenal and octanol (Table 3-1). Females emitted significantly higher amounts

of hexyl acetate, octyl acetate, (E)-2-octenyl acetate, (E)-2-octenal and octanol than

males (t test) (Table 3-1).

Table 3-1. Amounts of volatile compounds recorded in headspace of disturbed male and female Leptocorisa chinensis (n = 4)

Compound ng (relative amounts %) P value( t test)

Male Female Hexyl acetate 27±14 (0.5) 193±40 (1.2) 0.007 Octyl acetate 273±86 (5.5) 1170±347 (7.1) 0.046 (Z)-3-octenyl acetate 23±12 (0.4) 82±27 (0.5) 0.09 (E)-2-octenyl acetate 41±31 (0.8) 590±198 (3.6) 0.03 Octanal 22± 6 (0.4) 29± 3 (0.2) 0.31 (E)-2-octenal 4536±1191 (92.0) 14341±2208 (87.0) 0.008 Octanol 10±10 (0.2) 72±16(0.4) 0.02

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43

Response of L. chinensis to synthetic compounds of volatiles emitted from a

disturbed individual

I studied the responses of L. chinensis females to three volatile compounds, i.e.

octyl acetate, (E)-2-octenyl acetate and (E)-2-octenal, which were predominant in

volatiles emitted from a disturbed male and/or female (Table 3-1). The numbers of

individuals tested for each compound is shown in Fig. 3-3. Octyl acetate was the least

active in eliciting excitement/escape behaviour at two doses (Holm’s sequentially

rejective Bonferroni test after logrank test) (Fig. 3-3a, b). At 1-μg dose, no individuals

escaped from the arena when offered octyl acetate, while 60–90% of individuals

escaped when offered (E)-2-octenyl acetate or (E)-2-octenal with no significant

differences between the compounds (P = 0.29, log-rank test) (Fig. 3-3c). At the 10-μg

dose, the proportion of escape for octyl acetate was significantly lower than for the

other two compounds, while there were no significant differences between

(E)-2-octenyl acetate and (E)-2-octenal (Holm’s sequentially rejective Bonferroni test

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44

Undisturbed Undisturbed

Fig. 3-3. Proportions of excitement (a, b) and escape (c, d) of undisturbed Leptocorisa chinensis female when exposed to synthetic chemicals at different concentrations of (a, c) 1 μg and (b, d) 10 μg. Lines with the same lower-case letter are not significantly different (Holm’s sequentially rejective Bonferroni test after Kaplan–Meier time-to-event model using a log-rank test statistic, P<0.05). n = 19 for (E)-2-octenal, 17 for (E)-2-octenyl acetate, 7 for octyl acetate

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Concentration of (E)-2-octenal in air needed to induce escape behaviour in L.

chinensis

Males started showing flight behaviour when exposed to 0.001% solution of

(E)-2-octenal, while females started showing flight behaviour when exposed to 0.01%

solution (Fig. 3-4). The solution concentration significantly affected the flight behaviour

(F2,16 = 10.19, P = 0.001). However, effects of sex and the interaction (concentration 9

sex) were not significant (sex: F1,16 = 10.13, P = 0.72; interaction: F2,16 = 2.44, P = 0.30).

The headspace analyses of air in the cage showed that 0.01 and 0.1% solutions of (E)-

2-octenal resulted in 1.5 ppbV and 9.3 ppbV, respectively. The concentration of

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46

Discussion

Undisturbed males and females of L. chinensis became excited and escaped from

the arena when exposed to previously excited individuals of the same gender. Their

behaviours suggested that they responded to volatiles emitted from disturbed

conspecifics. Alternatively, visual/physical cues, such as walking and sounds associated Fig. 3-4. Proportions of flight behavior of undisturbed Leptocorisa chinensis males and female in mesh cage when offered emulsified (E)-2-octenal at different concentrations

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47

with movement, might have affected the excitation and escape behaviour. To clarify the

effects of the volatiles, we conducted chemical analyses and tested the effects of the

volatile components on the behaviour of L. chinensis.

Most of the volatiles found from the disrupted individuals were C8 aldehydes,

alcohol and acetates. The major compounds found in the headspace of disturbed males

and females were (E)-2-octenal and octyl acetate. (E)-2-Octenyl acetate was also one of

the major compounds in the headspace of disrupted females. Gunawardena and

Bandumathie (1993) reported that the chemical compositions of defensive secretions

produced by disturbed Leptocorisa oratorius males and females were similar; the two

major components in both were (E)-2-octenal (76% v/v) and octyl acetate (16%w/w). In

this study, in total, females emitted～3 times more volatiles than males. A similar trend

was reported in disturbed and undisturbed Lygus lineolaris (Wardle et al. 2003).

The relative amounts of (E)-2-octenal and (E)-2-octenyl acetate were 87–92% and

0.8–3.6%, respectively. Thus, we concluded that (E)-2-octenal was one of the major

factors eliciting the excitement and escape behaviour in L. chinensis females in the

arena when a disturbed individual of the same gender was introduced. No significant

differences in escape proportions were observed between males and females (Fig. 3-2b),

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48

escape behaviour in L. chinensis males. This conclusion was supported by the bioassays

using emulsified (E)-2-octenal in the mesh cage.

The proportions of escape in response to a disturbed female and to synthetic

volatile compounds [(E)-2-octenal and (E)-2-octenyl acetate (1-μg dose)] were not

significantly different (P = 0.23, log-rank test). The low activity of hexyl acetate

suggested that the location of the double bond at the (E)-2 position was more important

than the presence of the aldehyde group. Further studies are needed to evaluate this idea.

Further, we did not test the effects of minor compounds (less than ca. 1% in the blends

from both genders: hexyl acetate, (Z)-3-octenyl acetate, octanal and octanol) on

excitement and escape behaviours. Studies on the ecological functions of such volatiles

are needed as well.

Leptocorisa chinensis males are strongly attracted to a 5:1 mixture of

(E)-2-octenyl acetate and octanol (Leal et al. 1996; Watanabe et al. 2009; Fukatsu et al.

2012). These compounds were also detected from males and females at ratios of

approximately 2:1 and 20:1, respectively, in our study. Leal et al. (1996) also reported

that a whole blend did not attract males and the attractiveness of a binary mixture

decreased with addition of (Z)-3-octenyl acetate. In this study, we also detected

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49

(E)-2-octenyl acetate and octanol emitted from a disrupted adult, if any, would have

been hampered by the different ratios and/or by the presence of other compounds, such

as (Z)-3-octenyl acetate.

Leal et al. (1996) identified compounds found in this study as well as nonanal and

(E)-2-octenol in the headspace and hexane extracts of both male and female L. chinensis

that were anaesthetised with CO2 to minimize the release of defensive secretion.

CO2-induced anaesthesia might have resulted in production of small amounts of

volatile compounds. Our inability to detect volatiles from undisturbed males and

females was probably due to differences in collection methods.

As mentioned in the ‘‘Introduction,’’ in Sri Lanka, farmers protect rice plants by

putting smashed adult stink bugs, including Leptocorisa species, around their fields

(Yamashita, personal communication). Our data suggest that the volatiles from these

smashed bugs would have repelled stink bugs from the agricultural fields. In this study,

we clarified that (E)-2-octenal and (E)-2-octenyl acetate are involved in the

excitement/escape behavior of L. chinensis. Continual release of synthetic (E)-2- octenal

in paddy fields at concentration of ca. 2 ppbV or higher is expected to protect rice grains

from L. chinensis damage during critical stages of the growing season. I in chapter 4

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50

Chapter 4

Evaluation of the behavior regulator of Leptocorisa chinensis to control this species

in paddy fields

Introduction

The previous chapter outlines the possible use of volatile compounds eliciting

excitement/escape behavior in L. chinensis to control this species in paddy fields

(Chapter 3). The life history of L. chinensis has also been estimated to predict and

control L. chinensis invasion during the rice heading stage (Chapters 1 and 2).

However, to the best of our knowledge, the efficacy of the behavior regulator of L.

chinensis against the control of this species has not yet been investigated in paddy fields.

The aim of the present study was to establish a program for the pest control of L.

chinensis in paddy fields using a behavior regulator of L. chinensis. To achieve this goal,

we investigated the effectiveness of the behavior regulator of L. chinensis in paddy

fields.

We conducted behavior regulator release experiments in two open paddy fields at

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51

August and September 2006. The experimental details are summarized in Fig.4-1, We

conducted two release experiments, trials 1 and 2. Each experiment involved two plots,

one in which behavior regulators were released and a control plot that received no

treatment (Fig. 4-1). Two paddy fields were transplanted with seedlings at intervals of

18 to 20 days from mid-May. Each field had an area of 350–600 m2 and adjoined the

other, and the rice variety was Koshihikari. No insecticides were used in any of the

fields throughout the study. A fixed plot within each field of about 288 m2 (12 m × 24

m) was used for a routine population census. The routine population census was

conducted from August to September. The number of L. chinensis and peck rice were

systematically counted in 49 hills per plot on each census date. The concentration of the

behavior regulator was also estimated using Twister (Gerstel, M¨ulheim and der Ruhr,

Germany; 1 mm film thickness × 10 mm length), which uses magnetic stir bars coated

with polydimethylsiloxane, in each plot on each census date. The Twister was set as

shown in Fig.4-1. After one day, the Twister was taken to the laboratory and analyzed

by gas chromatograph–mass spectrometer (GC-MS).

To test for the effects of the number of L. chiensis and pecky rice on the behavior

regulator, the data were analyzed by t-test and one-way analysis of variance (ANOVA)

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52

Results and Discussion

The number of L. chinensis and the percentage of pecky rice in the plots with the

behavior regulator were lower than those in control plots (Figs. 4-2 and 4-3). In

particular, the number of pecky rice in each plot with the behavior regulator was

surprising less than 0.1, which is an excellent value for the investigation of rice grade

(Fig. 4-4). The concentrate of behavior regulator was recognized to be higher than the

threshold to release excitement/escape behavior in L. chinensis.

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53

Fig. 4-2 Number of N. chinensis in alarm pheromones and control sites.

P<0.05

N

o

.

o

f

L.c

h

in

en

si

s

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54

Based on these results for the open paddy fields, the behavior regulator would be

effective for the control of L. chinensis to reduce pecky rice. Thus, the utilization of the

behavior regulator can be considered a new technique for pest control. The behavior

regulator does not harm beneficial species and could become a key technique for

efficient and sustainable insect management strategies in the future.

1 2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 V2 V1 Control Alarm pheromones % of p ec ky ri ce P<0.05

Fig. 4-3 Percentage of pecky rice in alarm pheromones and control sites. Vales translate to arcsince.

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55 0 2000000 4000000 6000000 8000000 10000000 12000000 14000000 16000000 18000000 0 5 10 15 20 25 30 35 C once nt ra ti on of al ar m pher om one

Distance from alarm pheromones (m)

Fig. 4-4 Relationship between distance from alarm pheromones and concentration of them in a paddy field. Different letters indicate significant differences (P<0.05 Bonferroni corrections after ANOVA). Red line indicates the threshold.

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56

Summary

The rice stink bug Leptocorisa chinensis (Dallas) (Hemiptera: Alydidae) is a

major cosmetic pest, and is one of the main causes of pecky rice (Suzuki, 2001). Thus,

the effective and environmentally benign control of this species in paddy fields is

essential, without the need for chemical insecticides. The aim of the present study was

to develop a new method of control using a behavior regulator found in L. chinensis.

In Capters 1 and 2, I examined the life history of L. chinensis to predict and

control L. chinensis invasion during the rice heading stage. First, I investigated the

effects of a range of constant temperatures (18°C–30°C) on the development of eggs,

nymphs and the pre-oviposition period of adult females in L. chinensis. The duration of

all stages decreased as the temperature increased from 18°C to 30°C. Developmental

thresholds of 8.1°C, 10.1°C and 9.6°C were estimated for the eggs, nymphs and

pre-oviposition period, respectively. Thermal constants of 147 and 370 degree-days

above the thresholds of 8.1 degree-days and 10.1°C were required for the development

of the eggs and nymphs, respectively. The pre-oviposition period required 256

degree-days above the developmental threshold of 9.6°C. After placing individuals

collected in November and February in an incubator in the laboratory, 469.7

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57

to be equal to that of diapause female adults, were estimated to be required for the

pre-oviposition period of individuals that had overwintered. The present results were

useful to predict the field population phenology of L. chinensis in Japan.

Next, the effects of temperature and photoperiod on diapause induction and

termination in L. chinensis were studied under constant conditions or by using transfer

experimental protocols. Nymphs were reared either under a long-day (16:8 [L:D] h) or a

short-day (12:12 [L:D] h) photoperiod at 25°C in the laboratory. Females oviposited at

25°C and 20°C under the long-day photoperiod. However, females did not lay eggs

within 100 d at 15°C under the long-day photoperiod or at 20°C and 15°C under

short-day conditions. At 25°C, when nymphs were reared under a long-day photoperiod

and transferred to a short-day photoperiod on the day of adult emergence, females

started oviposition in 10 d but stopped shortly thereafter. When nymphs were reared

under a short-day photoperiod and transferred to a long-day photoperiod on the day of

adult emergence, females started oviposition in 40 d. Females that had been transferred

from the field on 1 February and 29 March to long-day laboratory conditions at 25°C

started oviposition in 40 d. However, females that had overwintered in the field were

transferred to a short-day photoperiod at 25°C on 1 February and 29 March did not start

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58

considered to be sensitive to reproductive diapause induction and termination signals

both before and after overwintering. Female L. chinensis require not only a long-day

photoperiod and 40 d at a temperature of 20°C or higher but also threshold temperatures

for the start of oviposition even after the winter. From the results of Chapters 1 and 2, I

was able to predict L. chinensis invasions during the rice heading stage.

In Chapter 3, I examined whether L. chinensis escaped from disturbed

conspecifics in an observation arena under laboratory conditions. When an undisturbed

individual of the same gender was introduced into the arena, the initial occupying L.

chinensis did not show any behavioral responses. However, when a disturbed

conspecific of the same gender was introduced, the initial occupant was immediately

excited and escaped from the arena through a hole, suggesting that the pungent volatiles

from the disturbed conspecific caused excitement/escape behavior.

Next in Chapter 3, I analyzed the volatiles emitted by both disturbed and

undisturbed L. chinensis and observed the responses of the undisturbed L. chinensis to

the components in the volatiles from disturbed conspecifics. Chemical analyses using a

GC–MS showed that disturbed adults of both sexes emitted octanal, (E)-2-octenal,

octanol, hexyl acetate, (Z)-3-octenyl acetate, octyl acetate and (E)-2-octenyl acetate.

近畿大学学術情報リポジトリ

博 士 学 位 論 文

Studies on development of the new method of control

using behavior regulators of Leptocorisa chinensis

(Dallas) (Hemiptera: Alydidae)

博 士 学 位 論 文

Studies on development of the new method of control

using behavior regulators of Leptocorisa chinensis

(Dallas) (Hemiptera: Alydidae)

Contents

Nymphs

Pre-ovispoition

n

d (mean±SD)

n

d (mean±SD) n

89

47.1 ± 1.9

81

29.5 ± 3.6

14

34

31.5 ± 1.7

29

21.1 ± 2.3

11

250

25.2 ± 2.5

142

17.3 ± 5.3

14

197 18.7 ± 2.1

172 12.4 ± 3.2

20

0

250

500

750

1000

J F M A M J J A S O N

D

Preoviposition period

Nymph

Egg

OW

G1

G2

G3

0

250

500

750

1000

J F M A M J J A S O N

D

Preoviposition period

Nymph

Egg

OW

G1

G2

G3

N

o

.

o

f

L.c

h

in

en

si

s

博士学位論文

博士学位論文

_{d (mean±SD)}

_n

_{d (mean±SD) n}

_G2

_G2